Food Lipids: Chemistry, Nutrition, and Biotechnology

at levels of 1–100 �g per component (up to 1.0 mg total) for routine separations.
Retention volumes of eluted sterols are usually expressed relative to cholesterol.
Gas–liquid chromatography (GLC) is most frequently used as an analytical
technique to monitor fractions during the isolation and separation of sterols; however,
this technique can also be used in preparative separations. The use of GLC as an
analytical and preparative technique for sterols and related steroids has been discussed
in numerous reviews [64,65]. The separation of sterols in gas–liquid systems
depends on the polarity and molecular weight (frequently correlating with size and
volume) of the molecule.
3. Characterization of Sterols
GLC is most frequently used to quantitate sterols in extracts, subfraction mixtures,
and isolated sterol fractions. Selectivity is provided by gas–liquid partitioning. In
general, three different methods of detection have been employed for quantitation:
flame ionization detection (FID), electron capture detection (ECD), and mass detection
(MD) [66,67]. However, FID is by far the most commonly employed method
because it is relatively insensitive to temperature changes during analysis and to
minor structural differences in sterols and related steroids, and because it has a large
linear mass range of response. For quantitative analysis it is necessary to determine
the linear range or response to a sterol standard (e.g., cholesterol or �-cholestanol)
for each FID and accompanying chromatographic system. For sample analysis, dried
fractions are routinely weighed and dissolved in a known amount of solvent to give
a mass-to-volume ratio within the linear range of the detector, whereupon the samples
are injected onto the column.
Quantification of sterols in extracts, subclass fractions, or isolated sterol fractions
by HPLC is somewhat limited. The selectivity provided by either adsorption
or reversed phase chromatography is in many cases greater than that provided by
GLC. Most frequently, UV detectors are employed and are set at end-adsorption
wavelengths [68]. These detectors have limited sensitivity, with monochromatic detectors
being most sensitive. Additionally, UV detectors cannot be considered universal
sterol detectors. Most sterols differ significantly in their UV absorption properties,
even in end-absorption regions. Thus, it would not be possible to select a
single wavelength for the quantitation of complex sterol mixtures. Therefore, HPLC
coupled to variable-wavelength detectors or multidiode detectors can be used to
quantitate specific sterols in a mixture if the compound in question has a unique
absorption spectrum relative to other members of the mixture.
For a full discussion of the identification of sterols with NMR [69,70], mass
[71], UV [72], infrared, and X-ray [73] spectrographic techniques, the reader is
referred to recent comprehensive reviews in this area.
REFERENCES
1. R. J. Hamilton. Commercial waxes: Their composition and application. In: Waxes:
Chemistry, Molecular Biology and Functions (R. J. Hamilton, ed.). The Oily Press,
Ayr, Scotland, 1995, p. 257.
2. A. H. Warth. Chemistry and Technology of Waxes. New York, Reinhold, 1956.
3. R. Sayers. Wax–An Introduction. European Wax Federation and Gentry Books, London,
1983.
Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.